The invention is designed to improve the medical care of patients with thrombotic or thromboembolic disease, such as deep venous thrombosis (DVT) and pulmonary embolism (PE), by facilitating clinical diagnosis and by providing a means by which the effectiveness of treatment can be measured.
The most challenging aspect of caring for patients with DVT and PE concerns making the initial diagnoses. In fact, most PE fatalities occur before the disease has been detected. Clinical signs and symptoms are neither sensitive nor specific; and the subsequent evaluative process for diagnosing DVT and/or PE is time consuming, expensive, and potentially invasive. The non-invasive diagnostic techniques for these diseases focus on demonstrating defects in the vascular anatomy, findings that are not specific for active thrombosis. For example, compression ultrasonography (CUS) can be used to detect pathology in the femoral vein. However, the distinction between a new thrombus and focal wall thickening from a previous thrombus cannot be made reliably. In addition CUS cannot be used to reliably detect asymptomatic DVT. As a result, attention has been focused on the identification of a serologic marker that would indicate active thrombosis.
In the last decade, the utility of plasma D-Dimer levels for identifying thromboembolic disease has been investigated extensively. D-Dimers are formed as a result of the degradation of cross-linked fibrin, and plasma levels have been shown to be elevated in both DVT and PE. However, plasma D-Dimer levels reflect the rate of fibrinolytic activity, but not necessarily the rate of fibrin formation. As a result, plasma D-Dimer levels are elevated in a variety of pathologic conditions involving previous fibrin formation, such as sepsis, DIC, pneumonia, and malignancy. In fact, only 22% of medical inpatients (presumably without thomboembolic disease) do not have elevated D-Dimer levels.
Although anticoagulants have been used for decades to treat thromboembolic disease, venous thromboembolism (VTE) in particular, the ideal method of treating this disease is unresolved. It is generally accepted that early anticoagulation dramatically reduces short-term mortality (Douketis et al. 1998), and it is becoming apparent that the incidence of long-term sequelae such as recurrent DVT and PE are also dependant upon the intensity of treatment in the first few days after diagnosis (Hull et al. 1997). The optimal method of early anticoagulation for VTE is, however, a controversial issue. New anti-thrombotic strategies are constantly in development, including improved dosing regimens for unfractionated heparin (Raschke et al. 1993, Lopaciuk et al. 1992, Hirsch et al. 1996), low molecular weight heparins (Levine et al. 1996, Koopman et al. 1996m Meyer et al. 1995) and specific inhibitors of the coagulation enzymes thrombin (Verstraete 1997) and factor Xa (Walenga et al. 1997). Each regimen has a specific anti-thrombotic potency, defined as its ability to suppress in situ thrombus propagation. It is likely that the benefits of these newer anticoagulant strategies will depend on the relationship between early anti-thrombotic effects and long-term clinical outcomes.
Although anticoagulants may have different mechanisms of action, the ultimate biochemical goal is the same, to prevent thrombin-mediated conversion of fibrinogen to fibrin and thus stop thrombus propagation (anti-thrombosis). Unfortunately, the anticoagulant potencies of these medications, measured by in vitro tests of activity such as the activated partial thromboplastin time (aPTT) and the plasma anti-Xa activity, do not reliably predict their anti-thrombotic effects in animal models (Carrier et al. 1993), Carrier et al. 1992, Morris et al. 1998).
There is growing recognition that inadequate initial treatment of VTE predisposes to fatal pulmonary emboli (Dalen 1986) and long-term recurrence (Hull 1997). However, there are limitations to the data suggesting that low antithrombotic activity itself in the early treatment of VTE leads to poor clinical outcomes. For example, anticoagulant activities of patients receiving unfractionated heparin are generally measured using the plasma aPTT, which has only a moderate correlation with actual plasma heparin levels (Gawoski et al. 1987, Brandt et al. 1981, van den Besselaar et al. 1990)). Furthermore, even the moderate correlation between anticoagulant activity and anti-thrombotic effect observed in animal models of thrombosis has not been validated in humans. Finally, the assumption that the intensity of anti-thrombosis correlates with the clinical efficacy, though reasonable, has not been tested in humans. For example, there have been no clinical studies to correlate VTE recurrence with the anti-thrombotic effects of anticoagulation.
Measuring the anti-thrombotic effects of anticoagulants in humans with VTE is difficult. The most commonly used non-invasive tests for diagnosis of DVT (compression ultrasonography, impedance plethysmography and magnetic resonance imaging) and PE (ventilation-perfusion scanning and helical CT scanning) do not provide sufficient anatomical information to determine reliably whether thromboemboli have enlarged acutely. Invasive studies such as contrast venography and angiography, while better at demonstrating gross changes in thrombus size (Lopaciuk et al. 1992, National Heart 1970) can be painful and are often impractical for following treatment. Furthermore, they may not be able to detect subtle increases in clot dimensions due to ongoing thrombosis. Finally, all of the anatomical tests described above share the limitation of being unable to differentiate the effects of anticoagulation (preventing clot enlargement) from the effects of the intrinsic fibrinolytic system (reducing clot size).
The most commonly used serological test for VTE, the D-dimer test, is also unsuitable as a marker of acute thrombosis. Although increasingly recognized as a sensitive indicator of VTE, the test measures thrombolytic fragments from pre-existing clots, and would not correlate with thrombus propagation. Likewise, serum markers of thrombin activation, such as prothrombin F1+2 fragments and thrombin-antithrombin III complexes, are not direct indicators of fibrin(ogen) conversion and polymerization. Thus anticoagulants with different spectra of activity against factor Xa and thrombin (for example heparin pentasaccharide and hirudin) would be expected to affect these tests differently, even if their in vivo and anti-thrombotic effects were the same.
Therefore, an ongoing need exists for a reliable test for DVT and PE, and also for a test to determine the effectiveness of different therapeutic regimens. Also the discovery of a marker with sufficient specificity and sensitivity in detecting PE and/or DVT would aid in diagnostic accuracy, and facilitate cost-effective utilization of resources. Thus, only those patients with a positive test would require anticoagulation and further evaluation with the appropriate tests.
The present invention provides a method for detecting thrombotic or thromboembolic disease, such as PE and/or DVT, by measuring the levels of fibrinopeptide B (FPB) in a physiological sample. The sample may be blood, plasma or, preferably, urine. The present invention also provides methods for monitoring the treatment of thrombotic or thromboembolic disease in a patient by monitoring changes in the levels of FPB in blood, plasma or, preferably, urine. The present invention also provides assay methods for conducting these measurements. The invention also provides peptides that include sequences from FPB; these peptides may be used as calibrators or controls in assays for FPB, they may be linked to carrier proteins and used to generate antibodies against FPB and/or they may be linked to labels or solid phases and used as competitors in competitive assays for FPB. The invention also provides reagents, compositions, and kits for carrying out immunoassays for FPB.
The present invention provides a fibronopeptide B (FPB) peptide defined by an amino acid sequence indicated in SEQ ID NO:1, and an FPB peptide defined by an amino acid sequence indicated in SEQ ID NO:2. These peptides may be covalently linked to a carrier molecule, such as keyhole limpet hemocyanin (KLH). Also these peptides and derivatives thereof may be attached to a substrate, such as a gel, hydrogel, resin, bead, magnetic bead, electrode, nitrocellulose, nylon filter, microtiter plate, culture flask, or polymeric material. The peptide may have a detectable moiety operably linked to it, and the detectable moiety may be a radionuclide, enzyme, specific binding pair component, colloidal dye substance, fluorochrome, reducing substance, latex, digoxigenin, metal, particulate, dansyl lysine, antibody, protein A, protein G, electron dense material, chemiluminescent substance, electrochemiluminescent substance, electroactive compound or chromophore.
The present invention also provides an antibody or fragment thereof that specifically recognizes an FPB peptide defined by an amino acid sequence indicated in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. Such a fragment may be an Fab, F(abxe2x80x2)2, or Fv fragment. The antibody or fragment thereof may be attached to a substrate, such as a gel, hydrogel, resin, bead, magnetic bead, electrode, nitrocellulose, nylon filter, microtiter plate, culture flask, or polymeric material. The antibody or fragment thereof may have a detectable moiety operably linked to it, and the detectable moiety may be a radionuclide, enzyme, specific binding pair component, colloidal dye substance, fluorochrome, reducing substance, latex, digoxigenin, metal, particulate, dansyl lysine, antibody, protein A, protein G, electron dense material, electrochemiluminescent substance, chemiluminescent substance or chromophore.
The present invention further provides a continuous cell line that produces an antibody that specifically recognizes a target peptide, wherein the target peptide is an FPB peptide defined by an amino acid sequence indicated in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. The cell line may be a monoclonal antibody cell line.
The present invention further provides an animal that produces polyclonal antibodies that specifically recognizes a target peptide, wherein the target peptide is an FPB peptide defined by an amino acid sequence indicated in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. The target peptide may be covalently linked to a carrier molecule. It may be keyhole limpet hemocyanin (KLH).
The present invention provides diagnostic method for detecting thrombotic or thromboembolic disease in a patient having the step of detecting the presence or amount of FPB in a sample such as a physiological fluid taken from the patient, to determine whether the patient has thrombotic or thromboembolic disease. The thrombotic or thromboembolic disease to be detected may be deep venous thrombosis (DVT) or pulmonary embolism (PE). The physiological fluid to be tested may be a fluid, such as blood or urine. Examples of techniques that can be used for the detection step include mass spectrometry, peptide sequencing, chromatography (e.g., HPLC or TLC), electrophoresis (e.g., capillary electrophoresis), enzyme-linked immunosorbent assay, immunonephelometry, agglutination, precipitation, immunodiffusion, immunoelectrophoresis, electrochemiluminescent immunoassay, electrochemical immunoassay, chemiluminescent immunoassay, western blot, immunofluorescence, radioimmunoassay, and immunohistochemistry. The amount of FPB present in the sample is considered xe2x80x9cpositivexe2x80x9d for thrombotic or thromboembolic disease if it is significantly above the normal range or if it is in a range that is indicative of thrombotic or thromboembolic disease. The exact cutoff values used will vary depending on the desired assay sensitivity and selectivity. In one embodiment, the amount of FPB present in a blood or plasma sample is considered xe2x80x9cpositivexe2x80x9d for thrombotic or thromboembolic disease if it is above 5 ng/ml, and in particular if it is above 10 ng/ml. In an alternative embodiment, the amount of FPB present in a urine sample is considered xe2x80x9cpositivexe2x80x9d for thrombotic or thromboembolic disease if it is above 50 ng/ml, and in particular if it is above 100 ng/ml.
The present invention provides diagnostic method for detecting thrombotic or thromboembolic disease in a patient having the steps of contacting a physiological sample suspected of containing fibrinopeptide B (FPB) and des-arginine FPB with an amount of detection agent specific for FPB to form an FPB:detection agent complex; wherein the detection agent is an antibody or fragment thereof that specifically recognizes an FPB peptide defined by an amino acid sequence indicated in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; and detecting the presence or amount of FPB:detection agent complex present in the sample to determine whether the patient has thrombotic or thromboembolic disease. The method may include a step of removing fibrinogen from the physiological sample. The thrombotic or thromboembolic disease to be detected may be deep venous thrombosis (DVT) or pulmonary embolism (PE). The physiological fluid to be tested may be a fluid, such as blood, plasma or urine. The detection step may be by enzyme-linked immunosorbent assay, immunonephelometry, agglutination, precipitation, immunodiffusion, immunoelectrophoresis, electrochemiluminescent immunoassay, chemiluminescent immunoassay, electrochemical immunoassay, western blot, immunofluorescence, radioimmunoassay, or immunohistochemistry. The amount of FPB:detection agent complex present in the plasma sample is considered xe2x80x9cpositivexe2x80x9d for thrombotic or thromboembolic disease if it is above 5 ng/ml, and in particular if it is above 10 ng/ml. The amount of FPB:detection agent complex present in the urine sample is considered xe2x80x9cpositivexe2x80x9d for thrombotic or thromboembolic disease if it is above 50 ng/ml, and in particular if it is above 100 ng/ml.
The present invention provides a method for monitoring the treatment of thrombotic or thromboembolic disease in a patient by monitoring changes in the levels of FPB in physiological samples such as blood, plasma, or ,preferably, urine. The monitoring may comprise the steps of contacting a physiological sample suspected of containing fibrinopeptide B (FPB) and des-arginine with an amount of detection agent specific for FPB to form an FPB:detection agent complex, wherein the detection agent is an antibody or fragment thereof that specifically recognizes an FPB peptide defined by an amino acid sequence indicated in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; detecting the amount of FPB:detection agent complex present in the sample; repeating the steps at a point later in time; and comparing the amounts determined at the two time points and correlating the change in the amounts to determine whether the thrombosis or embolism is diminishing in size. The method may include a step of removing fibrinogen from the sample.
The present invention also provides a diagnostic method for detecting thrombotic or thromboembolic disease in a patient involving contacting a urine sample suspected of containing fibrinopeptide B (FPB) and des-arginine FPB with an amount of detection agent specific for FPB to form an FPB:detection agent complex, wherein the detection agent is an antibody or fragment thereof that specifically recognizes an FPB peptide defined by an amino acid sequence indicated in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; detecting the presence or amount of FPB:detection agent complex present in the sample to determine whether the patient has thrombotic or thromboembolic disease.
Moreover, the present invention provides a diagnostic method for monitoring the treatment of thrombotic or thromboembolic disease in a patient by monitoring changes in the levels of FPB in the patients urine. Such monitoring may involve contacting urine samples suspected of containing fibrinopeptide B (FPB) and des-arginine FPB with an amount of detection agent specific for FPB to form an FPB:detection agent complex; wherein the detection agent is an antibody or fragment thereof that specifically recognizes an FPB peptide defined by an amino acid sequence indicated in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; and monitoring the changes in the urine concentration of FPB over time to determine if the thrombosis or embolism is diminishing in size.
The present invention also provides for kits that contain in one or more containers one or more of the reagents or compositions used in carrying out the assays of the invention. These kits may also contain calibration samples or standards.